Spectrofuorometerlab. 1

Fluorescence spectroscopy or (spectrofluorometry)

Is a type of electromagnetic spectroscopy which analyzes fluorescence from

a sample. It involves using a beam of light, that excites the electrons in

molecules of certain compounds and causes them to emit light.

fluorophore• A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical

compound that can re-emit light upon light excitation.

• Fluorophores typically contain several combined aromatic groups, or plane or cyclic

molecules with several π bonds.

• Fluorochromes absorb light of a particular wavelength, they are transiently excited and then

emit light of a longer wavelength as they return to their unexcited state.

• The emitted light is referred to as fluorescent light (fluorescence).

• The intensity of the emitted light is directly proportional to the amount of fluorochrome in

the sample.

• Not all the light energy that is absorbed by the fluorochrome is emitted as fluorescence

because some of the energy is quickly dissipated as heat or vibrational energy.

Instrument design

• The instrument design must provide some mechanism by which different

excitation and emission wavelengths of light are selected.

• This is achieved by use wavelength selectors which are

monochromators.

• Monochromators are device made of several components, one component

disperses the spectrum of visible light, while the other component is

adjustable, and is used to select the particular wavelength of light that

will pass through the monochromator .

Instrument design

• A spectrofluorometer must have an excitation monochromator and

emission monochromator because

• The absorption and emission spectrum of one compound differs from that of another compound.

• It should always be kept in mind that the wavelength selector must be adjusted for every compound tested.

Principle of spectrofuorometer function• The excitation light signals is visible light consisting of many wavelengths

radiating in all directions. Xenon arc lamp is often used as an alternative

source because

• A device known as excitation monochromators is used to focus the light onto a

prism or grating, which cause dispersion of the spectrum of light. Computer

driven exit slits determine the wavelength of the light that exits the

monochromators and enters the cuvette. it is crucial that all extraneous

wavelengths of the light be blocked before entering the cuvette because this

would contribute to the measured emission.

Principle of spectrofuorometer function• The excitation light penetrates the cuvette and is absorbed by the

fluorochrome, when the fluorochrome absorbs light energy it, it is excited

momentarily. As the fluorochrome reverts back to its more stable state,

light of longer wavelength is emitted. • Fluorescence is emitted in all directions. A slit in the emission

monochromator allow light to focused onto the wave selector. The

emission monochromator is set to the wavelength of light emitted as

the fluorochrome returned to its stable state.

Principle of spectrofuorometer function• The emission monochromator is spatially arranged at 90º to the

excitation monochromators to minimize the amount of excitation light

that reaches the detector .

• The detector is often a photomultiplier tube (electron tube) that absorbs

the emitted light and ejects electron in proportion to the amount of

light absorbed.

A schematic of spectrofluorometer

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Determination of unknown quinine sulphate

concentration using calibration curve

• In five 50 ml volumetric flasks, prepare a serial dilutions of quinine sulphate by pipette 2,4,6,8,10 ml from the stock solution of Q.S (5 g/L).

• Complete to volume with 0.2N H2SO4 and mix well.• Set the λ excitation at 350 nm and λ emission at 450 nm.• Record the relative fluorescence intensity for each dilution, and for the

unknown sample.• Plot the calibration curve by plotting relative fluorescence intensity versus

concentration.• Determine the unknown conc. of QS from the graph. • 0

procedure

ml that you pipette from quinine sulphate stockCalculate conc. of Q.S for each diluted flask

Stock Q.S concentration (5 g / L)

Relative Fluorescence

intensity

2mlC X V = C; X V5) g / L (X 2 ml = C; X 50 ml

? from the instrument

4mlC X V = C; X V5) g / L (X 4 ml = C; X 50 ml

? from the instrument

6mlC X V = C; X V5) g / L (X 6 ml = C; X 50 ml

? from the instrument

8mlC X V = C; X V5) g / L (X 8 ml = C; X 50 ml

? from the instrument

10ml C X V = C; X V5) g / L (X 10 ml = C; X 50 ml

? from the instrument

Unknown sample conc.? from the instrument

From the calibration graph

calculation

Unknown concentration

How to determine unknown concentration from the calibration curve

Determination of unknown quinine sulphate

concentration using linear equation

XY

10.260

20.4120

30.6180

40.8244

51310

1 -press AC …... Mode ….. (choose 3: STAT)…... (Choose 2: A +B X)

2- Fill the schedule with the calculated concentration in X column, and with fluorescence intensity obtained in Y column. As shown.

After each number you add

press = to transfer to the next row

3- press AC….. Shift 1 ….. Choose 7: Reg ……. (Then choose 1 : A) …. Press = ……. You will have intercept value.

4- press AC….. Shift 1 ….. Choose 7: Reg ……. (Then choose 2 : B) …. Press = ……. You will have slope value.

Y = a + bx

Unknown Conc.

slope

Unknown fluorescence intensity

intercept

Several factors affect the amount of fluorescence that is measured and so not all

emitted light is measured. The most common factor is quenching .

Quenching refers to a decrease in fluorescent intensity. Quenching by the

solvent may occur when the fluorochrome interacts with molecules in the

solution. A variety of processes can result in quenching such as :

energy transfer, complex-formation and collisional quenching.

The most common chemical quenchers are:

•Oxygen , iodide ions, chloride ions and acrylamide.

Factors that affect fluorescence

measurements

processes can result in quenching

Determination of the quenching effect of KI on the fluorescence of

quinine sulphate

procedure

• In eight 100 ml volumetric flasks, add 16 ml of Q.S.

• Add 0,1,1.5,2.5,5,7,10,15 ml of KI ( stock conc. 0.1M) to each flask.

• Complete to volume with distilled water.

• Read and record relative fluorescence intensity for each flask then plot intensity versus conc. of KI

• Comment on the curve

calculationml of Q.S.ml of KI Calculate conc. of KI for each diluted flask

))Stock conc. 0.1M

Intensity

160C X V = C; X V0.1 M X 0 ml = C; X 100 ml

?from the instrument

161C X V = C; X V0.1 M X 1 ml = C; X 100 ml

?from the instrument

161.5C X V = C; X V0.1 M X 1.5ml = C; X 100 ml

?from the instrument

162.5C X V = C; X V0.1 M X 2.5ml = C; X 100 ml

?from the instrument

165C X V = C; X V0.1 M X 5 ml = C; X 100 ml

?from the instrument

167C X V = C; X V0.1 M X 7 ml = C; X 100 ml

?from the instrument

1610C X V = C; X V0.1 M X 10 ml = C; X 100 ml

?from the instrument

1615C X V = C; X V0.1 M X 15ml = C; X 100 ml

?from the instrument

Effect of of KI on the fluorescence of quinine

sulphate

What is the difference between Fluorescence spectroscopy and spectrophotometer?